Methods for producing elastin, and tropoelastin products for repairing and or replacing tissue

ABSTRACT

The invention is directed to a method including the steps of providing at least one layer of unlaminated elastin or unlaminated elastin-based materials or unlaminated tropoelastin materials. Then, the unlaminated elastin or unlaminated elastin-based materials or unlaminated tropoelastin materials is subjected to heating and pressing steps. The pressing step of the present invention is preferably conducted in the presence of steam. The laminated elastin or laminated elastin-based materials or laminated tropoelastin materials preferably comprises a multi-layer composite material. Typically, the step of adhering with an adhesive material the laminated elastin or laminated elastin-based materials or laminated tropoelastin materials is employed in order to achieve a water-tight engagement with the tissue substrate. A biogradable cyanacrylate glue is generally used in order to achieve quick and easy way to secure the patch in place and provide watertight fusion instantly. Preferably, the adhesive material comprises an alkoxy alkyl cyanoacrylate material.

RELATED APPLICATIONS

This application claims benefit to provisional application No.60/136,573 filed May 28, 1999. This application is a 371 national stageapplication of PCT/US00/14621 filed May 25, 2000.

This invention was made with Government support under Grant No.DAMD17-96-1-6006 awarded by U.S. Army Medical Research AcquisitionActivity. The U.S. Government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates to laminated elastin, laminatedelastin-based biomaterials, and to laminated tropoelastin materials, tomethods of producing such laminated materials, and more particularly tomethods of using these laminated materials in tissue repair andreplacement.

BACKGROUND OF THE INVENTION

Elastin fibers are responsible for the elastic properties of severaltissues such as skin and lung, as well as arteries, and are composed oftwo morphologically distinct components, elastin and microfibrils.Microfibrils make up the quantitatively smaller component of the fibersand play an important role in elastic fiber structure and assembly.

The most abundant component of elastic fibers is elastin. The entropy ofrelaxation of elastin is responsible for the rubber-like elasticity ofelastic fibers. Elastin is an extracellular matrix protein that isubiquitous in mammals. Elastin is found, for example, in skin, bloodvessels, and tissues of the lung where it imparts strength, elasticityand flexibility. In addition, elastin, which is prevalent in theinternal elastic lamina (IEL) and external elastic lamina (EEL) of thenormal artery, may inhibit the migration of smooth muscle cells into theintima. Elastin in the form of solubilized peptides has been shown toinhibit the migration of smooth muscle cells in response toplatelet-derived factors (Ooyambia et al, Arter iosclerosis 7:593(1987). Elastin repeat hexapeptides attract bovine aortic endothelialcells (Long et al, J. Cell. Physiol. 140:512 (1989) and elastinnonapeptide s have been shown to attract fibroblasts (U.S. Pat. No.4,976,734). The present invention takes advantage of these physical andbiochemical properties of elastin.

Thirty to forty percent of atherosclerotic stenoses are opened withballoon angioplasty restenose as a result of ingrowth of medial cells.Smooth muscle ingrowth into the intima appears to be more prevalent insections of the artery where the IEL of the artery is ripped, torn, ormissing, as in severe dilatation injury from balloon angioplasty, vesselanastomoses, or other vessel trauma that results in tearing or removalof the elastic lamina. While repair of the arterial wall occursfollowing injury, the elastin structures IEL and EEL do not reorganize.Since these components play major structural and regulatory roles, theirdestruction is accompanied by muscle cell migration. There are alsodiseases that are associated with weakness in the vessel wall thatresult in aneurysms that can ultimately rupture, as well as other eventsthat are, at least in part, related to abnormalities of elastin.

In vertebrates elastin is formed through the secretion and crosslinkingof tropoelastin, the 72-kDa biosynthetic precursor to elastin. This isdiscussed, for example, in an article entitled “Oxidation, Crosslinking, and Insolubilization of Recombinant Crosslinked Tropoelastin byPurified Lysyl Oxidase” by Bedell Hogan, et al in the Journal ofBiological Chemistry, Vol. 268, No. 14, on pages 10345 10350 (1993).

In vascular replacement and repair, the best current option is toimplant autologous veins and arteries where the obvious limit is thesupply of vessels which can be sacrificed from the tissues they wereintended to service. Autologous vein replacements for damaged arteriesalso tend to be only a temporary measure since they can deteriorate in afew years in high pressure arterial circulation.

When autologous graft material is not available, a surgeon must choosebetween sacrificing the vessel, and potentially the tissue itsub-served, or replacing the vessel with synthetic materials such asDacron or Gore tex. Intravascular compatibility indicate that several“biocompatible polymers”, including Dacron, invoke hyperplasticresponse, with inflammation particularly at the interface between nativetissue and the synthetic implant. Incomplete healing is also due, inpart, to a compliance mismatch between currently used syntheticbiomaterials and native tissues.

As described in the prior co-pending patent applications assigned to theassignees of this application set forth above (patent application Ser.No. 08/798,426 filed Feb. 7, 1997, Ser. No. 08/797,770 filed Nov. 19,1998, Ser. No. 08/798,425 filed Feb. 7, 1997, Ser. No. 09/000,604 filedDec. 30, 1997, and U.S. Pat. No. 5,989,244, issued Nov. 23, 1999, andU.S. Pat. No. 5,990,379 issued Nov. 23, 1999) all of which areincorporated herein by reference, elastin and elastin-basedbiomaterials, or tropoelastin materials, can be used in a number ofmedical applications. For example, these materials can be employed toprovide a method of effecting repair or replacement or supporting asection of a body tissue, as a stent, such as a vascular stent, or asconduit replacement, or as an artery, vein or a ureter replacement, oras a stent or conduit covering or coating or lining. It can also providea graft suitable for use in repairing a lumen wall, or in tissuereplacement or repair in, for example, interior bladder replacement orrepair, intestine, tube replacement or repair such as fallopian tubes,esophagus such as for esophageal varicies, ureter, artery such as foraneurysm, vein, stomach, lung, heart such as congenital cardiac repair,or colon repair or replacement, or skin repair or replacement, or as acosmetic implantation or breast implant.

Surgical repair of major injury to the duodenum for example, withsignificant tissue los, requires innovative surgical techniques, and isassociated with significant morbidity and mortality. Segmental resectionand primary end to end anastomosis is not possible in this region due toits close proximity to the head of pancreas, and connections with commonbile duct and pancreatic duct. Small defects can be repaired by primaryclosure, which will result in stricture of the duodenum depending on theamount of tissue loss. Large defects cannot be repaired this way. Itwill require innovative techniques, such as creation of a Jejunal patch,duodenojejunostomy, serosal onlay patch, pyloric exclusion withgastrojejunostomy, or even pancreatico duodenectomy. The last procedureis fairly extensive, and is not likely to be tolerated by acutelyinjured patients with other multiple injuries. The first threeprocedures can be done, but they still require long surgery timeinvolving additional bowel anastomosis, and are feasible only when thejejunum is intact. Pyloric exclusion is accompanied by prolongedexternal drainage of the duodenal content, which makes it difficult tomanage fluid and electrolyte balance, and high incidence ofintraabdominal infection, sepsis and chronic fistula formation,predisposing the victim to prolonged intensive care, parenteralnutrition, hospitalization, and disability. This is due to the highcontent of electrolyte and digestive enzymes in the duodenal fluid,which comes mainly from bile and pancreatic excretion. As a result,prolonged leakage of duodenal content is associated with prolonged andextensive tissue loss and sepsis. Recent development in antibiotics andintensive care has significantly reduced the mortality rate from thiscondition but morbidity is still high.

SUMMARY OF THE INVENTION

The use of the subject laminated elastin and/or elastin basedbiomaterials and/or tropoelastin materials, typically in the form of aheterograft, which can employ a biodegradable glue or adhesive material,and which can be used for repair of defects such described above withrespect to the duodenom. The elastin and/or elastin-based biomaterialsand/or tropoelastin, in laminated form, can be used to provide areliable barrier to repair or replace a tissue substrate, typicallyinjured or diseased human tissue. The subject biodegradable glueprovides quick and easy water tight tissue fusion between the laminatedmaterials and the tissue substrate.

A technique has been developed that can easily, quickly, and reliablyrepair the injury to organs, such as the duodenum, without compromisingthe lumen. It should result in a much faster recovery with lesscomplications, morbidity, and mortality. The laminated elastin and/orelastin based biomaterials and/or tropoelastin can be used to repairsuch defect without compromising the lumen. Also, in the case of theduodenum there is no need for other bowel anastomosis, or extensiveresection. This laminated material was shown to be biologically inert,inducing minimal immunological response, and to be resistant toinfection, and hydrochloric acid.

More specifically, a method is provided herein for producing theabove-described laminated elastin or laminated elastin-based materialsor laminated tropoelastin materials. The laminated elastin or laminatedelastin-based materials or laminated tropoelastin materials is capableof being attached to a tissue substrate for repair or replacementthereof. The tissue substrate can comprise either a human or animaltissue.

The method comprises the steps of providing at least one layer ofunlaminated elastin or unlaminated elastin-based materials orunlaminated tropoelastin materials. Then, the unlaminated elastin orunlaminated elastin-based materials or unlaminated tropoelastinmaterials is subjected to heating and pressing steps. The pressing stepof the present invention is preferably conducted in the presence ofsteam. Preferably, depending on the thickness and nature of theunlaminated material and the desired laminated material finalproperties, the preferred pressure applied is from about 40 psi up toabout 2000 psi, and the preferred temperature during pressing is fromabout 50 degrees C. up to about 170 degrees C.

The laminated elastin or laminated elastin-based materials or laminatedtropoelastin materials preferably comprises a multi-layer compositematerial. More preferably, the laminated elastin or laminatedelastin-based materials or laminated tropoelastin materials arebilaminar pressed materials.

Typically, the step of adhering with an adhesive material the laminatedelastin or laminated elastin-based materials or laminated tropoelastinmaterials is employed in order to achieve a water-tight engagement withthe tissue substrate. A biogradable cyanacrylate glue is generally usedin order to achieve quick and easy way to secure the patch in place andprovide watertight fusion instantly. Preferably, the adhesive materialcomprises a cyanoacrylate material. More preferably, the cyanoacrylatematerial comprises an alkoxy alkyl cyanoacrylate material.

The method of this invention can also include the step of suturing thelaminated elastin or laminated elastin-based materials or laminatedtropoelastin materials to the tissue substrate. Preferably, the suturingstep is affected without substantial tearing of the laminated elastin orlaminated elastin-based materials or laminated tropoelastin materials.

Further objects and advantages of the invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.

FIG. 1 is a schematic representation of a laminated Elastin/SIScomposite patch of the present invention.

FIG. 1A is a photographical representation of a bilaminar Elastin/SIScomposite patch of this invention.

FIG. 2 is a photographic representation of a laminated Elastin/SIScomposite patch of FIG. 1.

FIG. 2A is a phographical representation of the laminated Elastin Patchof FIG. 1A as applied to a duodenum employing a polycyanoacrylate glue(Poly-Med GF-62) as the adhesive medium.

FIG. 3 is a schematic representation of the laminated Elastin/SIScomposite patch of FIG. 1 applied to an opening in a Duodenum.

FIG. 3A is photographic representation of a completed duodenal repair inwhich a composite laminated patch is glued and sutured in place, withoutlaceration, and then wrapped with omentum.

FIG. 4 is a photographic representation of the 2 cm opening in theduodenum and application of a glue as an adhesive.

FIG. 4A is a schematic representation of a laminated duodenal patchrepair employing laminated elastin sandwiched by SIS being digested inthe duodenum.

FIG. 5 is a photographic representation of a laminated Elastin/SIScomposite patch applied to the duodenum.

FIG. 5A is an endoscopic view of the laminated Elastin Patch 5 daysafter implantation.

FIG. 6 is a photographic representation of the Elastin/SIS compositepatch applied to the Duodenum, wrapped with the Omentum.

FIG. 6A is an upper GI contrast study, 2 weeks after implant of thelaminated Elastin Patch.

FIG. 7 is a chart showing average body weight of the experimentalanimals.

FIG. 7A is a photographic representation of a gross specimen of thelaminated Elastin Patch 3 months after implant.

FIG. 8 is an endoscopic view of the laminated duodenal patch repair site5 days after the implant.

FIG. 8A depicts the histology of the laminated Elastin Patch.

FIG. 9 is a photographic representation of a duodenum 2 weeks after arepair using the laminated Elastin Patch in which a Barium study wasconducted.

FIG. 10 is a photographic representation of the gross specimen of theduodenum three months after the repair.

FIG. 11 is a photographic representation of the histology of a healedrepair site after three months.

FIG. 12 is a photographic representation of the gross specimen of theduodenum, one week after the repair, the SIS and dyed glue inside thelaminated elastin having been digested.

FIG. 13 is a photographic representation of the histology of the repairsite of an animal 20 days after the repair.

FIG. 14 is a pictorial representation of shear tests of lap welds usingtissue adhesives on elastin aortic patches.

FIG. 15 is pictorial representation of the breaking strength of GF-62lap welds on pressed aortic heterografts in both neutral and alkalinesolutions.

FIG. 16 is a further pictorial representation of the breaking strengthof GF-62 lap welds on pressed aortic heterografts in both neutral andalkaline solutions.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Elastin-based biomaterials suitable for use in the present invention canbe prepared, for example, from elastin (e.g. from bovine nuchalligament), fibrinogen and thrombin as described by Rabaud et al (U.S.Pat. No. 5,223,420). (See also Aprahamian et al, J. Biomed. Mat. Res.21:965 (1987); Rabaud et al, Thromb. Res. 43:205 (1986); Martin,Biomaterials 9:519 (1988).) Such biomaterials can have associatedthrombogenic property that can be advantageous in certain types oftissue substrate repair. Elastin-based biomaterials suitable for use inthe invention can also be prepared from elastin and type III collagen,also as described by Rabaud and co-workers (Lefebvre et al, Biomaterials13(1):28-33 (1992). Such preparations are not thrombogenic and thus canbe used for vascular stents, etc. A further type of elastin-basedbiomaterial suitable for use in the present invention is prepared asdescribed by Urry et al (see, for example, U.S. Pat. Nos. 4,132,746 and4,500,700) (See also U.S. Pat. Nos. 4,187,852, 4,589,882, 4,693,718,4,783,523, 4,870,055, 5,064,430, 5,336,256). All of these issued patentsare incorporated into this application by reference. Elastin matrixesresulting from digestion of elastin-containing tissues (eg arteries) canalso be used. Digestion results in the removal of cells, proteins andfats but maintenance of the intact elastin matrix. The biomaterial usedwill depend on the particular application.

Elastin-based biomaterial of the invention prepared from soluble elastin(see Rabaud et al above) can be molded so as to render it a suitablesize and shape for any specific purpose. Molded biomaterial can beprepared as follows. Elastin (eg soluble elastin (MW 12-32,000 daltons)is washed and swollen in buffer. Fibrinogen or cryoglobulins (prepared,for example, according to Pool et al, New Engl. J. Med. 273 (1965 areadded to the swollen elastin, followed by thiourea, with or without aprotease inhibitor (such as aprotinin), and collagen. Thrombin is addedwith stirring and the resulting mixture is immediately poured into anappropriate mold. The mold is then incubated (for example, at 37 DegreesC.) while polymerization of the fibrin/elastin material is allowed toproceed, advantageously, for from between 15 minutes to 1 hour, 30minutes being preferred. The reaction can be carried out at temperaturesless than 37 Degrees C., but the reaction proceeds more rapidly at 77Degrees C. Heating the reaction to over Degrees C., however, can resultin denaturation of the thrombin. Cooling of the mixture while stirringallows more time for mixing to occur. For polymerization to occur, it isimportant to have calcium and magnesium in the buffer and to useundenatured thrombin.

Following polymerization in the mold, the resulting biomaterial can befurther cross-linked using gamma radiation or an agent such asglutaraldehyde (a solution of glutaraldehyde, formic acid and picricacid being preferred). When radiation is used, the samples are,advantageously, subjected to gamma-irradiation from a Cobalt-60 source.The amount of irradiation can range, for example, from 10 to 100 MRAD,with 25 MRAD being preferred. It has been shown that the amount ofgamma-irradiation can affect the strength of the material (Aprahamian,J.Biomed. Mat. Res. 21:965(1987).

Sheets of unlaminated biomaterial can be prepared that are of acontrolled thicknesses by using appropriate molds. Sheets of thisbiomaterial can be made in thicknesses ranging, for example, from 200microns to 5 mm. By way of example, a sheet suitable for use as anintestinal patch can range in thickness from 200 microns to 5 mm, withabout 2 mm being preferred. A patch requiring greater strength, such apatch for use in the bladder, is typically thicker. Arterial patches canbe thinner, e.g., 100 um-1000 um.

Biomaterial prepared from soluble elastic or insoluble elastin fragmentscan also be molded into tubular segments for example, by injecting thematerial into tubular molds. Crosslinkage of the elastin solutionpresent between the inner and outer tubes can be effected prior towithdrawal of biomaterial from the mold or after the tubes are removed.Tubular segments of different inner and outer diameters, as well as ofdifferent lengths, can be prepared using this approach by varying thediameters of the inner and outer tubes. A mold of this type can be madein virtually any size with the inner and outer tubes varying indiameter. A small tube can be used for a coronary arterial stent. Alarge tube of 1-5 inches in diameter can be made and used as anangularly welded patch for anastomosis of the small intestine or colon.Various molding techniques and molding materials can be used; theforegoing is merely an example.

As indicated above, biomaterial suitable for use in the presentinvention can be prepared from digests of tissue containing an elastinmatrix. Tissues suitable for use as a starting material include arteries(e.g. coronary or femoral arteries, for example, from swine), umbilicalcords, intestines, ureters, etc. Preferably, the matrix material is(derived from the species of animal in which the implantation is beingperformed so that bio compatibility is increased. Any method of removing(digesting away) cellular material, proteins and fats from the nativematrix while leaving the extracellular elastin matrix intact can beused. These methods can involve a combination of acidic, basic,detergent, enzymatic, thermal or erosive means, as well as the use oforganic solvents. This may include incubation in solutions of sodiumhydroxide, formic acid, trypsin, guanidine, ethanol, diethylether,acetone, t-butanol, and sonication. Typically, the digestion proceedsmore quickly at higher temperatures. The optimal temperature and time(of incubation depend on the starting material and digestive agent usedand can be readily determined.

The biomaterial of the invention, whether prepared from elastin powderor from tissues digests, is normally secured to existing tissuesubstrate. Various techniques for effecting that attachment can be used,including art-recognized techniques. For example, biomaterial can besecured using a tissue substrate welding energy source and an agent thatabsorbs energy emitted by that source. This technique is described indetail in the above referenced U.S. Patents and U.S. patent applicationwhich are assigned to the assignee herein. (All of which have beenincorporated herein by reference.)

The laminated elastin and/or elastin biomaterial and or tropoelastin aspreviously described herein can be employed as a patch material(“Elastin Patch”) for use in, for example, intestinal or colon repairswhich frequently do not heal well with current techniques, particularlywhen the patient has nutritional or other problems or when the patientis in shock, such as in the case of multiple gunshot wounds or otherabdominal injuries. The use of such an Elastin Patch can, for example,seal off intestinal contents and thereby reduce the likelihood ofperitonitis. The use of an Elastin Patch will be described in detailbelow. In addition, a patch can be used on a solid organ, such as theliver, when lacerations have occurred. Similarly, the biomaterial of theinvention can be used to repair or replace portions of the urinarysystem i.e., from the calyces of the kidney on down to the urethra. Thepatch can also be used to seal a defect in a cardiac chamber, such as anatrial septal defect, as well as bronchial or rectal fistulas. Thelaminated biomaterial can also be used as a cerebrovascular patch for ananeurysm. The laminated biomaterial can be sealed in place with targetedlaser fusion. For applications where direct exposure is not possible ornot desirable, a variety of catheter or endoscopic systems can beemployed to direct the laser energy to the target site.

The laminated elastin, elastin-based biomaterial or tropoelastin canalso be used to replace portions of diseased or damaged vascular ornonvascular tissue substrate such as esophagus, pericardium, lung plura,etc. The laminated biomaterial can also be used as a skin layerreplacement, for example, in burn or wound treatments. As such, thelaminated biomaterial serves as a permanent dressing that acts as ascaffolding for epithelial cell regrowth. The laminated biomaterial caninclude antibiotics, coagulants or other (drugs desirable for varioustreatments that provide high local concentrations with minimal systemicdrug levels. A drug can be incorporated into the biomaterial therebydecreasing the need for systemic intravenous or oral medications. Thelaminated elastin biomaterial can be deployed with a dye on the tissueside and then fused with the appropriate wavelength and laser energy.

Furthermore, a drug can be incorporated into the layer of elastin and/orelastin biomaterial and/or tropoelastin thereby decreasing the need forsystemic intravenous or oral medications. Also, photodynamic therapydrugs (“PDT”) which are activated with light can be employed herein.

Elastin Patch can be created from porcine aorta. After harvesting, itwas preserved in 80% ethanol for 72 hours, then it was fully digested bysoaking in 0.5M NaOH at 90 degrees centigrade with sonication. Thedigested aorta is cut into 4×4 cm patches. Two patches were then pressedtogether at 121 degrees centigrade for 15 minutes to form one bilaminarElastin Patch. The bilaminar Elastin Patch was then packaged andsterilized at 121 degrees centigrade for 15 minutes.

Biodegradable Cyanoacrylate Glue was provided by Poly-Med, Inc. ofAnderson, S.C. The glue comprises a Gel composition of MethoxypropylCyanoacrylate (“MPC”), and L1 (Copolymer of lactide, glycolide, andcaprolactone). Cyanoacrylate adhesives are described in U.S. Pat. No.5,350,798.

In a series of experiments, twenty four domestic pigs were anesthetized,intubated, and under sterile technique, underwent celiotomy. The secondportion of the duodenum was mobilized, and was brought out of the wound.A circular defect of 2 cm in diameter was made on the second portion ofthe duodenum, excising half of its circumference. A circular ElastinPatch with a diameter of 3 cm was sandwiched between 4×4 cm of SIS sheetusing biodegradable Cyanoacrylate glue (FIGS. 1, 2). This Patch wasplaced over the duodenal defect using the same glue (FIGS. 3, 4), and afew interrupted sutures were used to anchor the patch in place to avoidmigration of the patch in case the glue fails (FIG. 5). This was furthercovered by omentum to provide vascular supply to the area of repair(FIG. 6). Celiotomy was closed, anesthesia was withdrawn, and the pigwas extubated. The pigs were allowed to resume regular feeding soonafter surgery, typically within the first hour after extubation. Theywere followed by clinical observation, weight gain, endoscopic studies,and Barium Swallow studies. Two to five months after surgery, the pigswere sacrificed to obtain the specimen, which were submitted forhistological examination. No antibiotics or antacids were given aftersurgery. Animal experiments were conducted strictly following theguidelines set by the Institutional Animal Care and Use Committee of theOregon Health Sciences University.

Twenty four domestic pigs were anesthetized and underwent celiotomy. A 2cm circular defect was created at the second portion of the duodenum byscissors, excising half of its circumference. The Elastin Patch,combined with SIS was applied to cover the defect using biodegradablecyanoacrylate glue and a few sutures. It was then covered with omentum.

Animals were followed by weight gain, endoscopic evaluation, and upperGI Barium studies. After 2-5 months, animals were sacrificed to obtainspecimens. One failed in 3 days due to a technical problem, and onefailed in 20 days due to an abdominal abscess. All other 22 animals(22/24, 91.7%) did well, gaining weight. Early endoscopic studies showedan intact patch.

Upper GI studies showed varying degree of stenosis at the repair site at3-4 months. Sacrifice after 2-5 months showed complete healing of thedefect, and dissolved patch. One animal had to be sacrificed at threedays after surgery. The patch has already partially dehisced with freeleakage of the bowel content. This was most likely due to technicalproblem, failing to glue that area of the patch. Another animal had tobe sacrificed at three weeks after surgery. This animal developed anabscess confined to retroperitoneal space, due to what appeared to be aperforation through the repaired site. The duodenal defect was still twocentimeters in diameter, covered with thick fibrous tissue, which had aperforation leading into the abscess cavity. Fragments of Elastin wereobserved within the fibrous scar. The internal surface of the scar wascovered with a thin layer of regenerated mucosa. The remaining twentytwo animals did very well, giving the success rate of 91.7% (22/24).They were able to resume oral feeding within the first hour aftersurgery. They were gaining weight following the normal growth curve ofdomestic pigs (FIG. 7). One animal was sacrificed at seven weeks, one attwo months, eleven at three months, seven at four months, and two atfive months.

Endoscopic study was performed at one and two weeks after surgery. Thelaminated patch was easily identified in the second portion of theduodenum, occupying up to half its circumference. The duodenum waseasily inflatable by injecting air, easily allowing the endoscope topass through the area of repair, suggesting no mechanical obstruction(FIG. 8).

Upper GI contrast studies were also performed to evaluate the functionof the duodenum. Early after surgery (one to two weeks), the repair siteshowed flat stiffening, but no significant stricture or obstruction tothe passage of contrast (FIG. 9). Late studies (more than two monthsafter surgery) showed varying degree of stricture of the repair site.

Gross specimen of the sacrificed animals showed complete healing of therepair site with mucosal coverage as early as seven weeks, whichappeared like a healed ulcer. There were varying degrees of hypertrophiccircular tissue substrate around the center of healing, causingmechanical obstruction, which seems to start resolving after five months(FIG. 10).

Histology of the specimens after 7 weeks showed a completely healedduodenal wall with mucosal regeneration in the center (FIG. 11).Submucosal tissue substrate has also regenerated with incompleteregeneration of the muscular layer. Nerves were also found in the centerof the regenerated tissue substrate. Submucosal tissue substrate showedmarked hyperplasia around the regenerated tissue substrate, whichcorresponds to the hypertrophic circular tissue substrate on the grossspecimen. There was no remnant of the Elastin Patch or SIS identified inthe specimen.

Major duodenal injury with significant tissue loss is a serious injury,which is very difficult to manage. It requires innovative surgicaltechniques, and has high morbidity, as mentioned previously. In treatinga trauma victim with penetrating injury to the duodenum, which mostlikely has coexisting serious associated injuries, such as to liver,pancreas, great vessels, and other bowels, the chances of survival isexpected to improve when a surgeon can perform a more definitive andreliable repair in a short amount of time. Primary repair is feasibleonly in relatively small injuries. Duodenal exclusion technique stillleaves bile and pancreatic excretions to drain through the duodenaldefects causing chronic fistulas. The chronic fistulas will causeelectrolyte imbalance, indigestion due to non physiologic state of theGI tract, and possible intraabdominal abscess and sepsis. Although themortality has decreased recently, owing to improvement in intensive careand antibiotics, the morbidity and cost of care remains high, andpatients suffer prolonged disability. More definitive procedures such asPancreaticooduodenectomy are too extensive for most of the criticallyinjured patients, and will not be tolerated. Jejunal patch and duonenojejunostomy will require long operating times. It is feasible only whenthe victim has intact jejunum, and it involves additional bowelresection and anastomosis.

The duodenal laminated patch repair which was developed can be performedvery easily, quickly, and reliably. The Elastin Patch is designed toprovide a reliable barrier for one to two weeks while tissue substrategrowth and regeneration into SIS takes place. This new regeneratedtissue substrate will, in turn, serve as an effective barrier, whileremaining elastin is completely digested in four to six weeks. Becausethe subject Elastin Patch itself is biodegradable by digestive enzymesin the duodenum, the risk of immunological response and infectiouscomplication of the patch is minimal. SIS is well integrated in theregenerated tissue substrate, and eventually disappears. This material,which is almost pure collagen, is an excellent scaffold for tissuegrowth.

It is believed that the SIS inside the Elastin is completely digested byduodenal content within a few days (FIG. 12). Elastin on the other hand,is much more durable, and serves as an effective barrier for two to fourweeks.

The biodegradable cyanoacrylate glue was used in order to simplify theoperative repair and to provide an immediate watertight seal against theduodenal enzymes. One critical problem we faced with our pure ElastinPatch was that the material was quite fragile, lacking in mechanicalstrength. Although not impossible, it was quite technically demanding toplace a suture through this material without laceration. Placing thislaminated patch with glue made it much simpler and easier, and initialmechanical strength of the tissue fusion was quite satisfactory.

Two animals did not survive the long term experiment. One of them neverregained GI continuity after surgery, and had to be sacrificed afterthree days. There was a leak at the distal medial corner of the ElastinPatch, and this was most likely due to a technical failure. We eitherfailed to apply the glue in this corner, or failed to apply the ElastinPatch before the glue dried out. The original glue had no color and wastransparent, but later a dyed glue was used to visually identify theglued area. The other animal started to deteriorate two weeks aftersurgery, losing appetite and body weight. At sacrifice on the twentiethday, we found that the animal had developed a retro peritoneal abscess.The area of defect was covered with thick fibrous tissue substrate,which had a 3 mm hole connecting to the abscess cavity. Histology showedfragments of elastin in the fibrous scar, and the inner surface wascovered with thin regenerated mucosa (FIG. 13). This animal had arelatively large hole in the duodenum, and inadequate tissue fusionbetween the bowel edge and the patch was felt to be the cause of earlydehiscence.

The subject patch is virtually made of pure elastin without impregnationof any enzyme inhibitors or antibiotics. Preliminary study to test itsresistance against hydrochloric acid in vitro showed no degenerationafter one week. Duodenal content, however, is mixture of bile,pancreatic enzymes, hydrochloric acid, ingested food, andmicroorganisms. Pancreatic digestive enzymes, such as elastase, couldrapidly degenerate the Elastin Patch.

Whereas enzyme inhibitor, Elgin C, strongly inhibits the solubilizationof human aortic elastin by human pancreatic elastase, the efficacy ofthe inhibitor sharply decreases if elastase is reacted with elastin forprolonged time. Surgical experiments were typically performed in thirtyminutes from skin to skin. The actual time required for duodenal repairitself was less than fifteen minutes. No other bowel resection oranastomosis is required, and our animals can resume oral feedingimmediately after they recover from anesthesia. For trauma victims withpenetrating injury to the duodenum this is an ideal treatment in thatthe surgery time is minimal and recovery is fast, eliminating the needfor prolonged hospitalization with antibiotics and intravenoushyperalimentation.

A major concern with this repair was late stricture of the repair site.In our experience, it seems to heal by both regeneration andcontraction, leaving a mild to moderate degree of stenosis, but not assevere as one would expect from healing by contraction alone. The centerof the healed scar showed evidence of mucosal regeneration. There wasalways a circular ridge of hypertrophied mucosa or submucosal tissuesubstrate around the center, which also seems to contribute to themechanical constriction of the duodenal lumen. This was most prominentat three months, and the two animals after 5 months showed tendency ofresolution, although objective quantification was not possible.

It is assumed that this is caused by the early degeneration of the glue,which allows the glued edge of the duodenum to fall into the lumen,exposing this part to the duodenal content. This circular edge willundergo inflammatory changes, resulting in hypertrophied circular scar.The glue (GF 62) is composed of Methoxypropyl Cyanoacrylate (MPC), andL1 (Copolymer of lactide, glycolide, and caprolactone). MethoxypropylCyanoacrylate with other modifiers result in higher adhesive strengththan isobutyl cyanoacrylate when used to approximate soft tissues. Theuse of cyanoacrylate glue to repair the injury may be also contributingto resistance against infection.

The Elastin Patch provides a reliable barrier to repair major tissuesubstrate defect in the second portion of the duodenum. By combiningthis patch with SIS, we could obtain a fairly strong material, which isresistant to infection and digestive enzymes, while allowing the tissuesubstrate to heal from outside.

The modified cyanoacrylate glue provides quick and easy water tighttissue fusion for the patch. Further modification to prolong itsfunction over two weeks is desired, which is likely to reduce the degreeof stricture at the repair site.

Regarding tissue adhesive testing, porcine aorta heterografts of pureelastin were cut length wise and unfolded into a rectangular sheet oftissue substrate. This 5 cm×6 cm sheet was pressed between two glassplates, immersed in a water bath, and then placed in the autoclave at121 C. for 15 minutes. Each heat pressed sheet was cut down the middleinto two halves. The halves were then overlapped and glued togetherusing GF 62 adhesive. This was allowed to cure overnight in a moistenvironment. After curing, each glued sample was cut into multiple 1cm×6 cm strips using a razor blade (FIG. 16). These samples wereimmersed in either a bath of PBS as the control or Sodium Phosphate witha pH of 8.6 for a specified time and then pulled with the ChatillonV1000 past the breaking point. The strength was calculated as thebreaking load per glued area. Three samples were used for testing ateach time point for each pH condition.

The results of the shear strength tests are shown in FIG. 4. The controlsections in PBS were only performed for 4 days and show very littlechange in breaking strength. The sections in the alkaline solutionincreased in strength to a maximum at 95 hours and maintained thisstrength until the last time point at 230 hours. The alkalineenvironment of the duodenum is not causing the glue to degradeprematurely.

Studies in this segment focused on (1) preparing several candidateabsorbable cyanoacrylate based compositions (Compositions Nos. GF 70, GF72, GF 74, GF 76, and GF 77) with improved adhesive strength retentionfor screening of the different candidate formulations; (2) incorporatinga safe dye into the cyanoacrylate based tissue adhesives (CompositionsGF 71, GF 73, GF 75, GF 62D, GF 76D, GF 77D and GF 78D) withoutcompromising their shelf lie and in vivo performance; (3) preparing,reproducibly, sufficient amounts of most promising tissue adhesives forevaluation (Compositions Nos. GF 62, GF 62D and GF 72D); (4) preparationof a series of highly absorbable tissue adhesives with expected highcompliance as cured films for evaluation in low load bearingapplications (Compositions Nos. HD 1, HD 2, HD 3 and HD 4); and (5)incorporating ground elastin and chopped PGA fibers in absorbable tissueadhesives for use as a hemostatic bandage (Compositions Nos. HB 1, HB 2and HB 3).

Two new test methods, the nylon film and fabric cleavage test weredeveloped and compared with the goat skin test method previouslydeveloped. Of the three methods, the fabric cleavage test was identifiedas the most reliable screening method.

The shear tests are conducted on 1.0 cm×2.0 cm pressed porcine aorticElastin Patches. Each piece is overlapped by 0.5 cm as indicated in FIG.14. The tissue adhesive is allowed to cure for 3-4 hours at which timethe pieces are pulled apart on the Chatillon V1000. The flexibilitytests are conducted on a 1.0 cm×2.0 cm pressed porcine aortic ElastinPatch by pre stretching the piece and then coating one side with tissueadhesive. The tissue adhesive is allowed to cure for 3-4 hours at whichtime the pieces are pulled apart on the Chatillon V1000. The change inthe initial slope of the load/displacement curve is an indication of theeffect of the tissue adhesive on the elasticity of the Elastin Patch.The results of the shear strength tests is shown in FIG. 15. Theadhesives are of equal or greater strength than the SuperGlue, which wasused as a control. Both GF 51 and GF 62D had the smallest effect onflexibility, while SuperGlue greatly decreased the elasticity of theElastin Patch.

Bilateral vertical neck incisions were made in a 80 lb. domestic swine.A surgical dissection proceeded down to the level of the carotid andjugular veins. The jugular vein was retracted laterally and theunderlying carotid arteries were exposed. 5000 units of heparin weregiven intravenously and the right carotid artery was isolated proximallyand distally. Due to lack of adequate vascular clamps a hemostat wasused to occlude proximal carotid flow. An 4 cm section of the rightcarotid artery was excised and an appropriately sized tubular carotidheterograft was brought onto the field. A running continuous suture of7-0 Prolene was then used to secure the proximal anastomosis. The sutureline was secured and the distal portion of the elastin graft wasoccluded with finger pressure. The proximal carotid hemostat clamp wasthen released allowing the elastin graft to be fully pressurized. Thegraft itself tolerated the pressurization well, however at theanastomotic site, there was significant leakage from the needle holes.In addition after approximately 5 minutes the needle hole sites startedto break down, eventually leading to a complete circumferencial tear ofthe elastin graft at the anastomotic sight.

The carotid artery was reclamped and a second elastin graft brought ontothe field This graft was prepared by painting a thin coating of PS MPCcyanoacrylate glue (Poly-Med, Inc.) onto the outside of the graftcovering approximately 5 mm. of each end. The glue was allowed to curefor 2 minutes. The glue coated graft had much greater tear strengthwhich assisted anastomotic stabilization. Anastomosis was then completedwith running a continuous suture of 7-0 Prolene from the proximalcarotid artery to the treated elastin graft. Upon releasing the clampthe suture line held tight with minimal to no leakage. Again the elastingraft pressurized normally. The distal end of the graft was thensimilarly treated in a distal anastomosis was created to the distalcarotid artery with a running suture of 7-0 Prolene in a similar fashionto the proximal anastomosis. The hemostats were then removed and thegraft was allowed to perfuse. The graft remained pulsatile and perfusingfor over an hour, however as the Heparin wore off (and most likely dueto the proximal carotid arterial injury secondary to the hemostat) thecarotid artery proximal the graft began to thrombose. This is certainlydue to iatrogenic injury caused by the unavailability of the appropriatevascular clamp.

A scanning electron micrograph of the lumenal surface of the heterograftis depicted. A vigorous thrombgenic response is absent from the surfaceof the graft. Occasional platelets and scattered red blood cells withprotein adsorption and mild fibrin adhesion. The heterograt was easy tohandle, but was tactically difficult to deploy without the addition ofthe PS MPC glue which served as backing able to withstand the tearingforce of the sutures. Work is underway to develop a cross bilaminatedcarotid heterograft with increased tear strength using the heat pressingtechnology developed for the aortic heterografts.

Poly-Med Inc. (Anderson, S.C.) provided cyanoacrylate tissue adhesives(HAF) to evaluate using Elastin Patches to repair lung and liverlacerations. Fresh porcine lungs with trachea intact were trimmed ofextraneous tissue substrate and kept on ice. A puritan manualresuscitator was attached to the trachea and the lungs were inflatedwith air. A 1×3 cm resection of the right upper lobe was discovered HAF3 adhesive was dispensed from a micropipette around the circumference ofthe patch. The Elastin Patch was placed over the lesion, pressed gentlyinto place, and allowed to cure. After 2 minutes the lungs were inflatedand the right upper lobe was airtight. Next, a 1.5 cm incision, 1 cmdeep, was cut with a scalpel into the right lower lobe, which waspreviously fully inflated. Haf 3 tissue glue was applied to the surfaceof the lung with a micropipette and spread around the incision with aglass tube. An Elastin Patch was placed over the incision, pressedgently into place, and allowed to cure creating an airtight seal. TheElastin Patches were tested for leaks after three hours. Both ElastinPatches developed small air leaks which were attributed to the gluecracking from excessive brittleness.

Elastin Patches were glued to a porcine liver with HAF 3 tissue adhesiveto evaluate the feasibility of using an elastin liver patch to repairliver lacerations. A 1.5 cm incision, 1 cm deep, was made on the liver.HAF 3 tissue glue was applied to the surface of the liver via amicropipette and spread around the incision with a glass tube. A singlelayer heat pressed aortic elastin graft was placed over the lesion,pressed gently into place, and allowed to cure for 3 hrs.

Strips of Elastin Patches were glued to the serosa of porcine lung andthe surface of liver with HAF tissue adhesives and tested for adhesivestrength. Fifteen microliters of glue was deployed via a micropipetteonto the surface of lung and liver strips. A 1 cm wide strip of singlelayer heat pressed aortic elastin was pressed gently into place andallowed to cure for a minimum of 10 minutes. Bonded specimens weremounted on a uniaxial tension tester and pulled apart until bondfailure. Sample numbers were too small for statistical comparisonbetween glue prototypes. Average shear strengths were as follows: lungpatch=55.0+/18.0 grams (n=5); liver patch=92.0+/18.0 grams (n=3).

Experimental new surgical repair of highly morbid duodenal injury usingElastin Patch and Biodegradable Cyanoacrylate. Of all thegastrointestinal tract injuries, such as with the gunshot wound in thebattle field, loss of duodenal tissue substrate from its second portionis extremely difficult to repair, due to its anatomical structure withits close attachment to the head of the pancreas and connections withthe biliary tree and pancreatic duct, which precludes resection with endto end anastomosis. Unless the injury is a single small defect, simplerepair is not possible in this region. Primary suture closure of a largedefect will compromise the lumen of the Duodenum, creating stricture andobstruction.

Major loss of tissue substrate in this region requires innovativetechniques, such as:

1) Diverticularization of the duodenum

2) Duodenal exclusion with gastrojejunostomy

3) repair with roux en Y duodenojejunostomy

4) Pancreaticoduodenectomy (Whipple A procedure)

First two procedures are accompanied by prolonged external drainage ofthe duodenal content, which makes it extremely difficult to manage fluidand electrolyte imbalance, and high incidence of intra abdominalinfection, sepsis, and chronic fistula formation, predisposing thevictim to prolonged intensive care, hospitalization, and disability.This is due to the high content of electrolyte and digestive enzymes inthe duodenal fluid, which comes mainly from pancreatic juice and bile.The pancreatic exocrine enzyme, Trypsin is excreted in inactive form,Trypsinogen, which is then activated by duodenal mucosal Enterokinase.The activated Trypsin will further activate other pancreatic exocrineenzymes excreted in inactive form, such as, Chymotrypsin, Elastase,Kallikrein, and Carboxypeptidase A & B. As a result, prolonged leakageof duodenal content is associated with prolonged and extensive tissuesubstrate loss and sepsis. Recent development in antibiotics andintensive care has significantly reduced the mortality rate from thiscondition, but morbidity is still high.

The third procedure is feasible only for limited amount of tissuesubstrate loss that can be covered by the jejunum, plus it requiresanother bowel anastomosis. Prolonged procedure in an acutely injuredpatient, with multiple other problems, will increase the mortality. Thisis also feasible only when intact jejunum is available.

The fourth procedure is very extensive, including the excision of thehead of the pancreas, as well as in the common bile duct, and isindicated only when both common bile duct and pancreatic duct, as wellas the duodenum, are involved in the injury. Very often, in the case ofmajor trauma, the victim with an unstable condition from another injury,hypothermia or shock cannot tolerate this procedure.

The Elastin Patch developed is used to repair defects in the duodenumwithout compromising the lumen, need for other bowel anastomosis, orextensive resection. This material acts as a skelton of native tissuegrowth, such as duodenal mucosa and serosa, to eventually cover thedefect, while providing mechanical barrier for the bowel content toavoid fistula formation. The material is acid resistant in vitro, and ispotentially resistant to various digestive enzymes and infectiveorganisms.

A sacrificed animal showed excellent fusion of the patch after 30seconds. In order to evaluate the use of elastin heterografts in repairof duodenum with their exposure to duodenal contents, an Elastin Patchof elastin heterograft was placed on defect created at the secondportion of duodenum. An adult mini swine around 30 lbs. was sedatedafter 24 hours of starvation, intubated and underwent general anesthesiawith Isoflurane. One dose of prophylactic antibiotic was givenintramuscularly (Cefotetan 500 mg). The animal was placed in supineposition. The abdominal wall was sterilized with betadine and draped ina sterile fashion. A 5 cm long horizontal incision was made on rightupper quadrant, just below the right second nipple, then the abdominalwall muscle and fascia were divided using electrocautery, and theperitoneal space was entered. The second portion of the duodenum wasexposed just below the incision. A full thickness defect 2 cm indiameter was created at the center of the second portion of duodenumwith scissors, hemostasis was achieved using electrocautery, and defectwas closed with the elastin heterograft patch using a biodegradablecyanoacrylate. The area of Elastin Patch repair was covered with omentumusing a few interrupted sutures. Abdominal wall was closed in layers.Polysporin ointment was applied to the incision line.

Twenty four hours after the surgery, the pig recovered well, toleratingoral diet, with normal activity. Five days after surgery, the pigcontinued to eat well. The animal was inspected in the cage, and it wasencouraging to learn that all the food was consumed, with no evidence ofemesis, and a large amount of stool was present in the cage. The animalappeared to have been in full normal activity. An endoscope wasperformed to visualize the duodenum. The stomach was nearly empty after24 hours of fasting, suggesting good emptying. The patch was occupyingabout ⅓ of the circumference, and was found to be intact. The mucosaledge had minimal inflammatory reaction. The duodenum had widely patentlumen with no stricture. The duodenum was easily inflatable, and thereappeared to be no leak. Several photographs were obtained.

In order to evaluate the use of an Elastin Patch heterograft in therepair of a duodenum with exposure to duodenal contents, an ElastinPatch heterograft was placed on a defect created at the second portionof a duodenum. A domestic swine around 30 to 40 pounds was sedated afterfasting for 24 hours, intubated and underwent general anesthesia withIsoflurane. One dose of prophylactic antibiotic was given intravenously(Cefotetan 500 mg).

The animal was placed in the supine position. The abdominal wall wassterilized with Betadine, and draped in a sterile fashion. A 5 cm longhorizontal incision was made on the right upper quadrant, just above theright second nipple, then the abdominal wall muscle and fascia weredivided using electrocautery, and the peritoneal space was entered. Thesecond portion of the duodenum was exposed, mobilized, and brought outof the incision. A full thickness defect of 2 cm in diameter, which wasabout half of the circumference was created at the center of the secondportion of the duodenum with scissors. Hemostasis was achieved usingelectrocautery, and the defect was closed with the Elastin Patchheterograft patch using a bioabsorbable cyanoacrylate (GF-62).

The area of patch repair was covered with Omentum using a fewinterrupted sutures. The abdominal wall was closed in layers. Antibioticointment was applied to the incision line. No oral antibiotics or antiacids were given after surgery. The follow up was done by clinicalevaluation of the animal, as well as endoscopic and Barium Swallowstudies.

TABLE 1 Results BW #1 BS Case # Pig # Op date #1 Patch Glue SutureEndoscope date Status Outcome 1 GI 001 Jul. 1, 1998 35 PE GF-52 Yes 5,12 days n/a Sac 7/13 Deh Patch 2 741 Aug. 19, 1998 35 S/DE/S GF-62 Yes9/22 69 lbs alive 3 734 Aug. 26, 1998 50 S/DE GF-62 Yes 9/22 75 lbsalive 4 745 Sept. 3, 1998 48 S/DE/S GF-62 Yes alive 5 747 Sept. 3, 199846 S/DE/S GF-62 Yes alive 6 748 Sept. 3, 1998 43 S/DE/S GF-62 Yes alive7 767 Sept. 11, 1998 40 S/DE GF-62 Yes alive 8 751 Sept. 11, 1998 41S/DE GF-62 No Sac 9/18 Dehisced Patch 9 752 Sept. 11, 1998 48 S/DE GF-62No Sac 9/21 Dehisced Patch 10 779 Sept. 21, 1998 51 S/DE/S GF-62 Yesalive 11 778 Sept. 21, 1998 48 S/DE/S GF-62 Yes alive 12 777 Sept. 21,1998 41 S/DE/S GF-62 Yes alive 13 797 Sept. 21, 1998 43 S/DE/S GF-62 Yesalive 14 781 Sept. 25, 1998 44 S/DE/S GF-62 Yes alive 15 782 Sept. 25,1998 45 S/DE/S GF-62 Yes alive 16 783 Sept. 25, 1998 41 S/DE/S GF-62 Yesalive 17 784 Sept. 25, 1998 41 S/DE/S GF-62 Yes alive 18 776 Sept. 25,1998 47 S/DE/S GF-62 Yes alive 19 785 Sept. 28, 1998 40 S/DE/S GF-62 Yesalive 20 786 Sept. 28, 1998 38 S/DE/S GF-62 Yes alive 21 787 Sept. 28,1998 49 S/DE/S GF-62 Yes alive 22 788 Sept. 28, 1998 40 S/DE/S GF-62 Yesalive 23 738 Sept. 28, 1998 76 S/DE/S GF-62 Yes alive 24 789 Sept. 29,1998 43 S/DE/S GF-62 Yes alive 25 790 Sept. 29, 1998 46 S/DE/S GF-62 Yesalive 26 791 Sept. 29, 1998 39 S/DE/S GF-62 Yes alive 27 792 Sept. 29,1998 51 S/DE/S GF-62 Yes alive

Experiment in vivo was conducted with a single Elastin Patch, usingbioabsorbable cyanoacrylate glue from Poly-Med, Inc. (GF 52). 24 hoursafter the surgery, the pig recovered well, tolerating oral diet, withnormal activity. 5 days after surgery, the pig continued to eat well.The animal was inspected and all the food continued to disappear, withno evidence of emesis, and large amount of stool. The animal was in fullnormal activity. An endoscopic procedure was performed to visualize theDuodenum. The stomach was nearly empty after 24 hours of fasting, whichsuggested good emptying. The patch occupied about ⅓ of thecircumference, and was found to be intact. The mucosal edge had minimalinflammatory reaction. The duodenum had a widely patent lumen with nostricture. The duodenum was easily inflatable and there was no leak.

An endoscopic procedure was repeated to follow up. The Elastin Patch wasfound to be dehisced. The pig started vomiting that morning. The pig wassacrificed and found that the Elastin Patch was completely dehisced.Histology showed some mild degeneration of the Elastin Patch with notissue substrate in growth.

In Case 2, bioabsorbable cyanoacrylate glue (GF 62) was used with abilaminar pressed Elastin Patch to enhance the durability. To enhancetissue growth around the Patch, SIS patches were added. The pig startedeating soon after surgery, and resumed normal activity by the next day.A Barium Swallow study was performed after surgery. The animal weighed65 lbs., gaining 34 lbs. since surgery. There was no dilatation of theproximal duodenum. The area of Elastin Patch looked smooth, with goodmotility, without obstruction. There was no leak. There was an area justproximal to the Elastin Patch, that showed some difficulty in dilatingwith peristalsis, but eventually the Barium passed through. This mightindicate some kink of the proximal duodenum due to the adhesive. Theanimal continues to do well.

In Case 3, using the same glue and the SIS only on the outside of theElastin Patch, it was determined whether the Elastin Patch couldtolerate the duodenal digestive enzymes, and also to see the potentialfor tissue growth with SIS only on the outside. A Barium Swallow wasperformed after surgery. The animal weighed 75 lbs., gaining 25 lbs.since surgery. The Barium passed very easily through the area of thepatch, without any evidence of obstruction or leak. The pig is alive anddoing very well.

Cases 4, 5, 6 were conducted using the same procedures and materialswere identical to Case 2. After surgery, they are doing very well. Wewill perform a Barium Swallow tests were conducted and they are doingwell.

Case 7, 8 and 9 had an identical procedure as the Case 3, and are doingwell. Cases 8 and 9 had the same Elastin Patch (SIS on outside only) andthe glue. The entire procedure was performed only with the glue, withoutany sutures, except for the abdominal wound closure.

Unfortunately, these animals started having problems with feeding after6 to 7 days, and we sacrificed them on postoperative day 7 and 10. Case8 had a dehisced patch with a leak into the peritoneal space, causingintra abdominal abscess. Case 9 had a completely dehisced patch whichcontained a leak within the omentum, forming a large abscess, causingduodenal obstruction and massive gastric dilatation.

The shear tests are conducted on 1.0 cm×2.0 cm pressed porcine aorticElastin Patches. Each piece is overlapped by 0.5 cm as indicated in FIG.15. The tissue adhesive is allowed to cure for 3-4 hours at which timethe pieces are pulled apart on the Chatillon V1000. The flexibilitytests are conducted on a 1.0 cm×2.0 cm pressed porcine aortic ElastinPatch by pre stretching the piece and then coating one side with 25 mlof tissue adhesive. The tissue adhesive is allowed to cure for 3 4 hoursat which time the pieces are pulled apart on the Chatillon V1000. Thechange in the initial slope of the load/displacement curve is anindication of the effect of the tissue adhesive on the elasticity of theElastin Patch.

The results of the shear strength tests is shown FIG. 16. The adhesivesare of equal or greater strength than the SuperGlue, which was used as acontrol. Both GF 51 and GF 62D had the smallest effect on flexibility,while SuperGlue greatly decreased the elasticity of the Elastin Patch.

In order to evaluate the use of Elastin Patch heterograft in repair ofduodenum with their exposure to duodenal contents, a patch of elastinheterograft was placed on defect created at the second portion ofduodenum. Adult mini swine around 30 to 40 lbs. was sedated afterfasting for 24 hours, intubated and underwent general anesthesia withIsoflurane. One dose of prophylactic antibiotic was given intravenously.(Cefotetan 500 mg)

The animal was placed on supine position. Abdominal wall was sterilizedwith Betadine, and draped in sterile fashion. A 5 cm long horizontalincision was made on right upper quadrant, just above the right secondnipple, abdominal wall muscle and fascia was divided usingelectrocautery, and peritoneal space was entered. The second portion ofthe duodenum was exposed, mobilized, and brought out of the incision. Afull thickness defect of 2 cm in diameter was created at the center ofthe second portion of duodenum with scissors, which was about half ofthe circumference, hemostasis was achieved using electrocautery, anddefect was closed with the Elastin Patch heterograft using bioabsorbablecyanoacrylate from Poly-Med, Inc.

The area of patch repair was covered with Omentum using a fewinterrupted sutures. Abdominal wall was closed in layers. AntibioticOintment was applied to the incision line. No oral antibiotics or antiacids were given after surgery. The follow up was done by clinicalevaluation of the animal, Endoscope, and a Barium Swallow contrast studyusing 27 implants were performed.

An experiment in vivo with single Elastin Patch, using Poly-Med glue.(GF 52). Within 24 hours after the surgery, the pig has fully recovered,tolerating oral diet, with normal activity. Endoscopic examination wasperformed to visualize the Duodenum. The stomach was nearly empty after24 hours of fasting, suggesting good emptying. The Elastin Patch wasoccupying about ⅓ of the circumference, and was found to be intact. Themucosal edge had minimal inflammatory reaction. The Duodenum had widelypatent lumen with no stricture. The Duodenum was easily inflatable, andthere was no leak.

A repeat endoscopic exam was performed as follow up. Unfortunately, thepatch was found to be dehisced. The animal care staff reported that thepig started vomiting from that morning. We sacrificed the pig and foundthat the Elastin Patch was completely dehisced. Histology showed somemild degeneration of the Elastin Patch with no tissue in growth.

Twenty six implants were performed. A bioabsorbable Poly-Medcyanoacrylate glue (GF 62) was used with a bilaminar pressed ElastinPatch to enhance the durability. To enhance tissue growth around thebilaminar Elastin Patch, we sandwiched it with small intestinalsubmucosa (SIS) patches. Also, in order to reduce the chance ofmechanical trauma to the Elastin Patch, we decided not to endoscoperoutinely. All the pigs started eating soon after surgery, and resumednormal activity by the next day. Only glue was used for applying theElastin Patch, without any sutures. These animals, however, had to besacrificed at 7 and 10 days after surgery due to dehiscence of theElastin Patch, with leak and abscess formation.

Of the remaining 24 animals, there were two early failures. One of themhad a leak on the 3^(rd) day, and had to be sacrificed. This was mostlikely due to a technical problem. The other one had to be sacrificed onthe 20th day due to a perforated patch. The cause of this is not quitecertain. All other 22 animals survived long term. Only one animaldeveloped a wound infection. In accordance with the animal careguideline, we electively sacrificed at 7 weeks. This animal showed noleak or obstruction of the duodenum, and the area of the patch hascompletely healed. Barium swallow tests were performed at 1 month aftersurgery. All the animals were gaining weight. The area of Elastin Patchlooked smooth, with good motility, without obstruction. There were noleaks.

Thirteen animals were sacrificed between 2 and 4 months. The remaining 8animals will be sacrificed between 4 and 5 months. All of the long termsurviving animals are showing some degree of stenosis at the area of thehealed duodenum, where the Elastin Patches were placed. The patchdisappears within 7 weeks, with complete healing of the defect. Thecenter of the defect shows coverage with mucosal cells, withhypertrophic changes around it. The future sacrifice at 4 months and 5months will provide us with more information about how these findingsare going to change. To better understand the mechanism of the healingprocess short term experiments were conducted, sacrificing 6 additionalanimals sequentially over 5 weeks. The repair technique designed formajor duodenal injury, employed an Elastin/SIS Patch and bioabsorbablecyanoacrylate glue. Late stenosis formation at repair the site is ofmajor concern, and we should continue with our effort to improve thisproblem through better understanding of the healing process.

Studies in this segment: focused on (1) preparing several candidateabsorbable cyanoacrylate based compositions (Compositions Nos. GF 70, GF72, GF 74, GF 76, and GF 77) with improved adhesive strength retentionfor screening of the different candidate formulations by OMLC; (2)incorporating a safe dye into the cyanoacrylate based tissue adhesives(Compositions GF 71, GF 73, GF 75, GF 62D, GF 76D, GF 77D and GF 78D)without compromising their shelf lie and in vivo performance uponevaluation; (3) preparing, reproducibly, sufficient amounts of mostpromising tissue adhesives for evaluation at OMLC (Compositions Nos. GF62, GF 62D and GF 72D); (4) preparation of a series of highly absorbabletissue adhesives with expected high compliance as cured films forevaluation in low load bearing applications at OMLC (Compositions Nos.HD 1, HD 2, HD 3 and HD 4); and (5) incorporating ground elastin andchopped PGA fibers in absorbable tissue adhesives for use as ahemostatic bandage (Compositions Nos. HB 1, HB 2 and HB 3).

Two new test methods, the nylon film and fabric cleavage test weredeveloped and compared with the goat skin test method previouslydeveloped. Of the three methods, the fabric cleavage test was identifiedas the most reliable screening method. The experimental protocol wasshared with the OMLC for use in their own screening studies.

Twenty-four domestic pigs were anesthetized and underwent celiotomy. A 2cm circular defect was created at the second portion of the duodenum byscissors, excising half of its circumference. Our Elastin Patch,combined with SIS was applied to cover the defect using biodegradablecyanoacrylate glue and a few sutures then it was covered with omentum.Animals were followed by weight gain, endoscopic evaluation, and upperGI studies. After 2-5 months, animals were sacrificed to obtainspecimens. One failed in 3 days due to a technical problem, and onefailed in 20 days due to an abdominal abscess. All other 22 animals(22/24, 91.7%) did well, gaining weight. Early endoscopic studies(5-14d) showed an intact patch. Upper GI studies showed varying degreeof stenosis at the repair site at 3-4 months. Sacrifice after 2-5 monthsshowed complete healing of the defect, and dissolved patch. The subjectElastin Patch material provides a reliable barrier to repair duodenalinjury and the biodegradable glue provides quick and easy water tighttissue fusion for our patch.

In an additional example, Elastin Patch was created from porcine aorta.After harvesting, it was preserved in 80% ethanol for 72 hours, then itwas fully digested by soaking in 0.5M NaOH at 90 degrees centigrade withsonication. The digested aorta is cut into 4×4-cm patches. Two ElastinPatches were pressed together at 121 degrees centigrade for 15 minutesto form one bilaminar patch. The Elastin Patch was then packaged andsterilized at 121 degrees centigrade for 15 minutes.

SIS patch was made from fresh porcine swine intestinal segment. Thetunica serosa and tunica muscularis were abraded by longitudinal wipingmotion with a scalpel handle and moistened gauze. The remainingintestine is everted, and tunica mucosa was removed by the same mannerof abrasion. The tube was then inverted to its original orientation.This was rinsed with saline several times and placed in 10% Neomycinsulfate solution and stored at 4 degrees centigrade. Decellularizationwas done by immersing the SIS in 2 mM SDS solution stirring withmagnetic bar for 1.5 hours. This was then washed in the 0.01 MIPBS (pH7.0) for 5 minutes, which was repeated 3-4 times. Finally, it was soakedin 0.01 M PBS (pH 7.0) with 10% Neomycin for storage.

Biodegradable cyanoacrylate glue was again provided by Poly-Med, Inc.,Anderson, S.C. The glue composition was 95% Methoxypropyl Cyanoacrylate(MPC), and 5% L1 (Copolymer of lactide, glycolide, and caprolactone).

Twenty-four domestic pigs were anesthetized, and under steriletechnique, underwent celiotomy. A circular defect of 2 cm in diameterwas made on the second portion of the duodenum, excising half of itscircumference. A circular Elastin Patch with a diameter of 3 cm wassandwiched between 4×4 cm of SIS sheet using biodegradable cyanoacrylateglue (See FIG. 1A. ). This composite Elastin Patch was adhered over theduodenal defect using the subject glue (See FIG. 2A), and a fewinterrupted sutures. This was covered by omentum and sutured to providevascular supply to the area of repair (FIG. 3A).

Animals were allowed to resume regular feeding soon after surgery. Noantibiotics or antacids were given after surgery. One animal had to besacrificed at three days after surgery due to a technical problem.Another animal had to be sacrificed at three weeks after surgery due toan abscess confined to retroperitoneal space. The remaining twenty-twoanimals did very well, giving the success rate of 91.7% (22/24). Theywere able to resume oral feeding within the first hour after surgery.They were gaining weight following the normal growth curve of domesticpigs. One animal was sacrificed at seven weeks, one at two months,eleven at three months, seven at four months, and two at five months.Endoscopic study was performed at one and two weeks after surgery. Thebilanior Elastin Patch was easily identified in the second portion ofthe duodenum, occupying up to half its circumference and there was nomechanical obstruction (FIG. 5A). Upper GI contrast studies showed flatstiffening, but no significant stricture or obstruction to the passageof contrast (FIG. 6A). Gross specimen of the sacrificed animals showedcomplete healing of the repair site with mucosal coverage as early asseven weeks, which appeared like a healed ulcer (FIG. 7A). Histology ofthe specimens after 7 weeks showed a completely healed duodenal wallwith mucosal regeneration in the center (FIG. 8A). Submucosal tissue hasalso regenerated with incomplete regeneration of the muscular layer.Nerves were identified, but no ganglion ingrowth was seen. There was noremnant of the elastin or SIS identified in the specimen.

Unlike conventional repairs, the Elastin Patch duodenal patch of thepresent invention can be performed very easily, quickly, and reliably.The subject Elastin Patch was designed to provide reliable barrier forone to two weeks while tissue growth and regeneration into SIS takesplace. The biodegradable cyanoacrylate provides an immediate water tightseal against the duodenal enzymes. Typically the surgical experiment wasperformed in thirty minutes from skin to skin. No other bowel resectionor anastomosis is required, and the animals can resume oral feedingimmediately after they recover from anesthesia. Healing occurs by bothregeneration and contraction, leaving mild to moderate degree ofstenoisis, but not as severe as one would expect from healing bycontraction alone. The center of the healed scar showed evidence ofmuscosal regeneration. There was always a circular ridge ofhypertrophied muscosa or submuscosal tissue around the center which isbelieved to be caused by the glue. In conclusion, the new Elastin Patchmaterial provides a reliable barrier to repair major tissue defect inthe second portion of the duodenum by combining this patch with SIS, afairly strong material, resistant to infection and digestive enzymes canbe obtained, while allowing the tissue to heal from outside. Themodified cyanoacrylate glue provides quick and easy water tight tissuefusion for the Elastin Patch of the invention.

I claim:
 1. A method for joining at least one layer of pressedbiomaterial to a tissue substrate, comprising: providing the pressedbiomaterial consisting essentially of elastin or tropoelastin materials;applying a biodegradable cyanoacrylate adhesive to either one of thematerial and the tissue; and adhering the pressed biomaterial to thetissue and forming a substantially water-tight engagement therebetween.2. The method of claim 1, wherein pressing is conducted at a pressure ofat least about 40 psi.
 3. The method of claim 1, wherein pressing isconducted a temperature of at least about 50 degrees C.
 4. The method ofclaim 1, wherein a suture can be effected without substantial tearing ofthe biomaterial.
 5. The method of claim 1, wherein the cyanoacrylateadhesive comprises an alkoxy alkyl cyanoacrylate adhesive material. 6.The method of claim 1, wherein the tissue substrate comprises humantissue.
 7. A method for producing a pressed biomaterial, comprising:providing at least one layer of an unpressed biomaterial consistingessentially of elastin or tropoelastin; and heating and pressing theunpressed biomaterial to form the pressed biomaterial which is capableof being attached to a tissue substrate for repair or replacementthereof.
 8. The method of claim 7, wherein the pressed biomaterialcomprises a multi-layer composite material.
 9. The method of claim 7,wherein a suture can be effected without substantial tearing of thepressed biomaterial.
 10. The method of claim 7, which further includesthe step of adhering with an adhesive material the pressed biomaterialin water-tight engagement with the tissue substrate.
 11. The method ofclaim 10, wherein the adhesive material comprises a cyanoacrylatematerial.
 12. The method of claim 11, wherein the cyanoacrylate materialcomprises an alkoxy alkyl cyanoacrylate material.
 13. The method ofclaim 7, wherein the tissue substrate comprises human or animal tissue.14. The method of claim 7, wherein the pressed biomaterial is abilaminar pressed biomaterial.
 15. The method of claim 7, which furtherincludes the step of suturing the pressed biomaterial to the tissuesubstrate.
 16. The method of claim 7, wherein the pressing step isconducted in the presence of steam.
 17. The product of claim 1, whichcomprises a pressed and steamed biomaterial.
 18. The method of claim 7,wherein pressing is conducted at a pressure of at least about 40 psi.19. The method of claim 7, wherein pressing is conducted at atemperature of at least about 50 degrees C.
 20. A biomaterial productwhich comprises at least one layer of a pressed biomaterial consistingessentially of elastin or tropoelastin biomaterial which is capable ofbeing attached to a tissue substrate for repair or replacement thereof.21. The product of claim 20, wherein the pressed biomaterial comprises amulti-layer composite material.
 22. The product of claim 20, which canreceive a suture without substantial tearing of the pressed biomaterial.23. The product of claim 20, which can be adhered with an adhesivematerial, the adhered biomaterial being in water-tight engagement withthe tissue substrate.
 24. The product of claim 23, wherein the adhesivematerial comprises a cyanoacrylate material.
 25. The product of claim24, wherein the cyanoacrylate material comprises an alkoxy alkylcyanoacrylate material.
 26. The product of claim 20, wherein the tissuesubstrate comprises human tissue.
 27. The product of claim 20, whereinthe pressed biomaterial is a bilaminar pressed biomaterial.